Fig 2: Study design and workflow. (a) Schematic representation of the study design and workflow. Time-zero kidney biopsy samples (pre-TCMR(b)) and POD-9 protocol biopsy samples (TCMR(b)) were obtained from two KT recipients. PBMCs were prospectively collected from six KT recipients before and after transplant. Kidney biopsies were subjected to spatial transcriptomics analysis using the GeoMx DSP platform, targeting a comprehensive panel of RNA biomarkers. PBMCs were labeled and processed for scRNA-seq using the 10X Genomics platform. (b) The UMAP plot shows the clustering of a total of 12 PBMCs samples. A total of 48,228 cells were annotated into 10 clusters, each representing distinct cell types characterized by specific surface markers. (c) Dot plot represents the gene expression profiles of key markers across the identified PBMC clusters. The size of the dot corresponds to the percentage of cells expressing a given marker within the cluster, whereas the color intensity reflects the average expression level of the marker in that cluster. (d) Comparative histological and immunofluorescent images of time-zero and POD-9 kidney biopsy samples. Histology sections underwent PAS staining, and selected ROIs were analyzed by GeoMx DSP for spatial gene expression. The fluorescence panel illustrates the cellular composition within the graft, using the following morphologic markers: nuclei stained in blue, CD45+ immune cells in green, PanCK+ epithelial cells in yellow, and α-SMA+ myofibroblasts in red. BDR borderline rejection, CTRL control, DSP digital spatial profiling, KT kidney transplantation, PBMC peripheral blood mononuclear cell, POD post-operative day, ROI region of interest, scRNA-seq single-cell RNA sequencing, TCMR acute T-cell mediated rejection, UMAP uniform manifold approximation and projection.